Immune cell recptor ligand and immune cell receptor

ABSTRACT

The present invention relates, in general, to immune cell receptor ligands and immune cell receptors. More specifically, the invention relates to an NKG2D immunoreceptor ligand and to an immune cell receptor having the same C-type lectin structure as the NKG2D receptor, and to nucleic acid sequences encoding same.

This application claims priority from Provisional Application No.60/408,397, filed Sep. 4, 2002, and Provisional Application No.60/478,371, filed Jun. 13, 2003, the contents of these ProvisionalApplications being incorporated herein by reference.

TECHNICAL FIELD

The present invention relates, in general, to immune cell receptorligands and immune cell receptors. More specifically, the inventionrelates to an NKG2D immunoreceptor ligand and to an immune cell receptorhaving the same C-type lectin structure as the NKG2D receptor, and tonucleic acid sequences encoding same.

BACKGROUND

Induction of tumor-specific cytotoxic lymphocytes (CTLs) by tumorantigens presented in the context of class-I MHC molecules and theactivation of natural killer (NK) cells play a critical role inantitumor immune response (Yee and Greenberg, Nat. Rev. Cancer2(6):409-419 (2002), Pardoll, Science 294(5542):534-536 (2001)). Thebalance between activating and inhibitory ligands expressed in tumorsmay critically affect the function of effector lymphocytes and theefficacy of antitumor immune response.

NKG2D is expressed on most CD8⁺ T-cells, γδ T-cells and NK cells andserves as one of the most potent activating receptors for effectorlymphocytes in peripheral tissues (Bauer et al, Science285(5428):727-729 (1999), Groh et al, Nat. Immunol. 2(3):255-260(2001)). NKG2D polypeptides associate with the adaptor molecule DAP10(and DAP12 in the mouse), providing a costimulatory signal to CD8⁺lymphocytes, or a primary stimulatory signal to NK cells, respectively(Diefenbach et al, Nature 413(6852):165-171 (2001), Jamieson et al,Immunity 17(1):19-29 (2002), Lanier et al, Nature 391(6668):703-707(1998)). NKG2D ligands mediate destruction of virus-infected cells andmark tumor cells for cell-mediated killing (Bauer et al, Science285(5428):727-729 (1999), Cosman et al, Immunity 14(2):123-133 (2001)).For example, in the mouse, ectopic expression of the NKG2D ligands Rae1bor H60 in tumor cell lines has resulted in cell-mediated rejection oftumors (Diefenbach et al, Nature 413(6852):165-171 (2001), Cerwenka etal, Proc. Natl. Acad. Sci. USA 98(20):11521-11526 (2001)). Furthermore,skin-associated NKG2D⁺ γδ T-cells successfully mediate destruction ofcarcinoma cells in vivo, utilizing a mechanism dependent on NKG2Dreceptor engagement (Girardi et al, Science 294(5542):605-609 (2001)).

Several human and murine molecules related to class-I majorhistocompatibility complex (MHC) molecules have been identified asligands for NKG2D. In humans, the ligands for NKG2D fall into either theMIC group or the UL16-binding protein (ULBP) group. The MIC groupconsists of MICA and MICB, which are closely related. Both are encodedwithin the human MHC locus and are expressed on a wide range ofepithelial tumors. MICA and MICB are stress-inducible molecules whichtrigger NK cell activation and function as costimulatory ligands thatcan substitute for B7 ligands (Bauer et al, Science 285(5428):727-729(1999)). The ULBP group consists of ULBP1, ULBP2 and ULBP3, which wereidentified based on their ability to bind to the human cytomegalovirusglycoprotein UL16 (Cosman et al, Immunity 14(2):123-133 (2001)).

The present invention results, at least in part, from thecharacterization of a tumor-associated MHC-I related ligand for theNKG2D receptor, designated herein as Lymphocyte Effector cell ToxicityActivating Ligand (Letal). Letal acts as a costimulator in CD8⁺ CTLs,inducing their expansion and activation. Letal also induces cytotoxicityin NK cells. Another embodiment of the invention results from theidentification of an immune cell receptor having the same C-type lectinstructure as the NKG2D receptor.

SUMMARY OF THE INVENTION

The present invention relates generally to immune cell receptor ligandsand immune cell receptors. More specifically, the invention relates toan NKG2D receptor ligand (designated “Letal”). The invention furtherrelates to an immune cell receptor that has the same C-type lectinstructure as the NKG2D receptor. The invention additionally relates tonucleic acid sequences encoding the ligand and the receptor.

Objects and advantages of the present invention will be clear from thedescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F. Letal is a new NKG2D ligand exhibiting a cytoplasmicdomain. FIG. 1A, The genomic sequence of Letal reveals the presence of 4exons separated by 3 short introns, the sequences of the 4 exons beingset forth in FIG. 1E (SEQ ID NOs:1-4) and a depiction of the translationof the cDNA into the amino acid sequence being set forth in FIG. 1F.FIG. 1B, Alignment of Letal (SEQ ID NO:8) and ULBPs (SEQ ID NOs:5-7).The transmembrane segment, spanning amino acids from 226 to 248, ismarked with asterisks. FIG. 1C, Phylogeny of all the murine and humanNKG2D ligands so far characterized, generated with the topologicalalgorithm of ClustalW. FIG. 1D, Letal is a ligand for NKG2D. All theresults shown are representative of at least 3 experiments. Left:Letal-transduced K562 cells exhibit a much stronger staining by apolyclonal anti-Letal Ab than mock-transductants. Incubation withGPI-specific Phospholipase C results in slight increase in binding,suggesting a positive effect in exposing epitopes, and confirming thetransmembrane structure of Letal. Shaded: Letal⁺ transductants; openthick: Mock-transductants; dotted: Treatment with Phospholipase C.Center, right: Control or Letal-transduced K562 and SKOV3 cells wereincubated with a NKG2D-Fc protein, and stained with anti-human Ig mAb.Shaded: Letal⁺ transductants; open thick: Mock-transductants. WhenLetal⁺-K562 cells are treated with GPI-specific Phospholipase C, sNKG2Dbinding decreases, but it is still stronger than that of mocktransductants, corresponding to the abundant expression of GPI-anchoredNKG2D ligands in K562 cell line (dotted). No significant decrease isobserved with SKOV3.

FIGS. 2A and 2B. Expression of Letal and NKG2D in different normaltissues and tumor cell lines. FIG. 2A, Letal is expressed by a varietyof normal tissues, as revealed by TaqMan analysis. FIG. 2B, total RNAfrom several ovarian and colon cancer cell lines was subjected to RT-PCRusing specific primers for Letal. The specificity of the products wasconfirmed by sequence analysis.

FIGS. 3A-3C. Regulation of Letal and NKG2D in tumor cells andlymphocytes. FIG. 3A, analysis of the effect of viral infection andinflammatory mediators on Letal expression in ovarian carcinoma cellsA2008. FIG. 3B, Retinoic acid treatment induces a progressive decreasein Letal mRNA expression. Results are representative of 3 experiments.FIG. 3C, up-regulation of NKG2D by fresh peripheral blood lymphocytesupon Letal engagement. Shaded: Unstimulated CD8⁺ cells.

FIGS. 4A-4C. Effects of Letal on CD8⁺ lymphocytes. Data arerepresentative of at least 3 experiments performed. FIG. 4A, Triggeringof peripheral CD8⁺ T-cells with K32-bound anti-CD3 results in strongerproliferation in the presence of Letal or anti-CD28, as measured by [³H]thymidine incorporation. FIGS. 4B, 4C, Combined triggering with anti-CD3and Letal or CD28 induces significant differences in IL-2 and IFN-γsecretion by CD8⁺ T-cells compared to anti-CD3 signaling alone.

FIGS. 5A and 5B. Expression of Letal induces the killing of cancer cellsby CD8⁺ and NK cells. FIG. 5A, Letal induces anti-tumor lymphocytecytotoxicity. Letal alone shows a modest effect in redirectingcytotoxicity against Letal⁺ K32 erythroleukemia cells by peripheral CD8⁺T-cells activated for 3 days with anti-CD3/Letal. Addition of anti-CD3mAb (0.5 μg/ml) increases specific lysis (x=effector:target ratio). FIG.5B, Ectopic expression of Letal increases NK cell-mediated cytotoxicityof the Letal⁻, p53⁻, chemoresistant ovarian cancer cell line SKOV3.

FIGS. 6A-6F. Immunohistochemical staining of advanced human ovariancarcinomas. Nuclei were counterstained with hematoxylin. These imagesare representative of different ovarian carcinomas, showing: FIG. 6A,high frequency of CD45⁺ leukocytes stained with anti-CD45 mAb(magnification: X20); FIG. 6B, high proportion of NKG2D+lymphocytes inmost specimens analyzed (magnification: X10); FIG. 6C, a high frequencyof tumor-infiltrating CD8⁺ cells is noted; in average, these cellsrepresented 15% of total leukocytes (magnification: X20) FIG. 6D, CD57⁺NK cells are only occasionally present (less than 1% of total CD45⁺cells), indicating a predominant role of T-cell-mediated responsesagainst advanced carcinomas; FIGS. 6E, 6F, expression of Letal in tumorislets of stage III ovarian carcinomas. Letal stainin is also noted ontumor-infiltrating leukocytes.

FIGS. 7A-7D. Letal and GLPD1 expression in normal and neoplastic ovariantissues. FIG. 7A, Quantification of Letal mRNA levels by TaqMan in humannormal ovaries and benign tumors (n=6); borderline tumors (n=4); stage I(n=9), and stage III ovarian carcinomas (n=29). FIG. 7B, Letal mRNAexpression analyzed by TaqMan PCR in tumor islets isolated by lasercapture microdissection. 12 specimens were evaluated with CD3⁺ cellsinfiltrating tumor islets and 7 with no T-cells in tumor islets. FIG.7C, Kaplan-Meier curves for the duration of overall survival, accordingto the presence or absence of Letal mRNA in 38 patients with stage IIIepithelial ovarian cancer. Letal expression was analyzed by Real-TimePCR. P values were derived with the use of log-rank statistic. FIG. 7D,Quantification of GLPD1 mRNA levels by TaqMan in the same specimens.Results are expressed as number of copies of the gene of interest pereach 10⁶ GAPDH copies.

FIGS. 8A-8C. Letal induces a sustained expansion of tumor-inflitratingCD28⁻ effector lymphocytes. FIG. 8A, Most CD8⁺ lymphocytes in solidtumors and ascites do not express the costimulatory molecule CD28. Gateon CD8⁺ cells. FIG. 8B, Sustained expansion of sorted CD8⁺ CD28⁻lymphocytes through CD3/Letal engagement. Left, tumor-inflitratinglymphocytes; right, lymphocytes sorted from tumor ascites. FIG. 8C,Combined triggering with anti-CD3 and Letal induced a dramatic increasein IFN-γ secretion by CD28⁻CD8⁺ 5 days after the third cycle ofstimulation. Results are compared to a pool of supernatants from thesame cells activated with anti-CD3 signaling alone.

FIGS. 9A-9C. Letal engagement protects lymphocytes fromcisplatin-induced apoptosis. FIGS. 9A, 9B, Letal stimulation inducesGlut-1 and increases glucose uptake by CD8⁺ lymphocytes. Letal⁻ K32cells were used as a non-stimulatory control. Shaded: Lymphocytesstimulated with CD3-alone. Total cellular Glut-1 was measured by flowcytometry; glucose uptake was evaluated with [³H]-2-deoxyglucose. FIG.9C, Letal engagement protects CD8⁺ lymphocytes from genotoxic drugs.Peripheral CD8⁺ T-cells were stimulated for 3 days with the indicatedfactors and then incubated with cisplatin. Results are expressed aspercentage of apoptotic cells. All the results are representative of atleast 3 experiments.

FIGS. 10A-10C. Letal engagement protects lymphocytes from Fas-dependentapoptosis. FIG. 10A, Selected ovarian carcinoma specimens exhibitintense FasL staining in cells by immunohistochemistry. FIG. 10B,Downregulation of Fas by peripheral blood lymphocytes upon CD3/Letalengagement. Lymphocytes were stimulated for 4 days with the indicatedconditions, and Fas expression was analyzed by flow cytometry. Shaded:Unstimulated CD8⁺ cells. FIG. 10C, Letal stimulation induces resistanceto FasL-dependent apoptotic death. CD8⁺ lymphocytes treated with theindicated factors for 4 days were exposed to anti-Fas Ab that deliversan apoptotic signal to Fas-sensitive cells. More than 25% ofLetal-stimulated lymphocytes resist apoptosis after 18 h. Arepresentative analysis of 3 experiments is shown. Results are expressedas percentage of non-apoptotic cells.

FIG. 11. cDNA sequence of immune cell receptor LCCR (SEQ ID NO:9).

FIG. 12. Predicted protein sequence of immune LCCR cell receptor (SEQ IDNO:10).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates, in one embodiment, to an MHC-1 relatedligand for the NKG2D receptor, and fragments and variants thereof, andto nucleic acid sequences encoding same. The ligand, which provides acostimulatory signal to CD8⁺ lymphocytes (inducing their proliferationand activation), is upregulated in certain tumors and inducescytotoxicity in NK cells.

In a specific embodiment, the ligand is a polypeptide having the aminoacid sequence set forth in SEQ ID NO:8 (see also FIGS. 1B and 1F), whichpolypeptide is referred to herein as “Letal”. This same polypeptide isreferred to as “ULBP4” by Chalupny et al (Bioch. Biophys. Res. Commun.305:129 (2003)). The invention includes this specific polypeptide andvariants thereof, as well as fragments thereof. The invention alsoincludes analogs/derivatives of such sequences.

Letal variants of the invention include polypeptides substantiallyidentical to the sequence of SEQ ID NO:8. Letal variants of theinvention do not include ULBP1, 2 or 3. The variants can include one ormore deletions, insertions, or substitutions relative to the sequence ofSEQ ID NO:8 (e.g., substitutions of one or more of the amino acids ofSEQ ID NO:8 wherein the substitution is with a conserved ornon-conserved amino acid (preferably, a conserved amino acid)). Thevariant can have an amino acid sequence that is, for example, at least50%, at least 60% or at least 70% identical to the sequence of SEQ IDNO:8, at least 80% identical, least 90% identical, at least 95%identical, at least 98% identical, at least 99% identical, or at least99.9% identical to the sequence of SEQ ID NO:8. The percent identity canbe determined, for example, by comparing sequence information usingBLAST 2 SEQUENCES. Variants in which differences in amino acid sequencerelative to the sequence of SEQ ID NO:8 are attributable to geneticpolymorphism (allelic variation among individuals producing the protein)are within the scope of the invention. Preferred variants include theresidues at the positions bolded in FIG. 1B that are common to Letal andthe depicted ULBP sequences.

Fragments of the invention include, but are not limited to,peptides/polypeptides comprising the signal peptide (e.g., about aminoacid 1 to about amino acid 28 of the sequence of SEQ ID NO:8), the α-1domain (e.g., about amino acid 29 to about amino acid 116 of thesequence of SEQ ID NO:8), the α-2 domain (e.g., about amino acid 117 toabout amino acid 207 of the sequence of SEQ ID NO:8), the transmembranedomain (e.g., about amino acid 226 to about amino acid 248 of thesequence of SEQ ID NO:8), the cytoplasmic domain (e.g., about aminoacids 249 to 263 of the sequence of SEQ ID NO:8) of the Letalpolypeptide of SEQ ID NO:8, or variants thereof. The invention alsoincludes fragments of the polypeptide of SEQ ID NO:8, preferably,fragments comprising at least 5 consecutive amino acids, morepreferably, at least 10 or at least 20 consecutive amino acids of thesequence of SEQ ID NO:8, or variant thereof. It will be appreciated thatfragments of the invention can be employed as immunogens, in generatingantibodies (monoclonal and polyclonal) using standard techniques.

Variants and fragments of the invention include, but are not limited to,polypeptides that retain a biological activity of the Letal polypeptide,for example, the ability to bind NKG2D receptor. An example of such apolypeptide is a soluble fragment of the sequence of SEQ ID NO:8, orvariant thereof. Such soluble polypeptides include, but are not limitedto, polypeptides comprising about amino acid 29 to about amino acid 225of the sequence of SEQ ID NO:8, or variant thereof. Polypeptides of theinvention can be tested for the ability to bind the NKG2D receptor inany suitable assay, such as a conventional binding assay. To illustrate,the polypeptide can be labeled with a detectable reagent (e.g., aradionuclide, chromophore, enzyme that catalyzes a colorimetric orfluorometric reaction, etc.). The labeled polypeptide can be contactedwith cells expressing NKG2D receptor. The cells can then be washed toremove unbound labeled polypeptide, and the presence of cell-bound labelcan be determined by a suitable technique, chosen according to thenature of the label.

In certain aspects, the sequences of GenBank accession numbers AY054974and AF359243 may not be within the scope of the invention.

The invention also includes derivatives/analogs of the Letal polypeptideof SEQ ID NO:8 and variants and fragments thereof. For example, theinvention includes polypeptides in which one or more of the amino acidresidues is fused with another compound, such as a compound to increasethe half-life of the polypeptide (for example, polyethylene glycol). Theinvention also includes glycosylated polypeptides, cyclic polypeptidesand polypeptides bearing a detectable label and/or bound to a solidsupport. The polypeptides can also include tumor-binding moieties, thatis the invention includes chimeric molecules (e.g., bispecific) thatinclude a Letal domain and an antibody variable domain directed againsta tumor specific epitope (e.g., folate binding protein or CA125 (ovariantumors)). The polypeptides of the invention can also be present as afusion protein, for example, to facilitate detection or isolation.

The invention includes isolated and purified, or homogeneous,polypeptides, both recombinant and non-recombinant. The polypeptides canbe synthesized chemically using art recognized techniques. Thepolypeptides can be used as described below or can be used in theproduction of antibodies (polyclonal or monoclonal) using standardtechniques. The invention includes such antibodies, and binding portionsthereof, as well as their use, for example, in detecting the presence ofa polypeptide of the invention in a sample (in which case, the antibodycan bear a detectable label).

The invention further relates to nucleic acid sequences encoding thesequence of SEQ ID NO:8, or variants, and fragments thereof, or thecomplements of such encoding sequences. One specific nucleic acidsequence encoding the amino acid sequence of SEQ ID NO:8 is that setforth in SEQ ID Nos:1-4 (see also FIGS. 1E and 1F). DNAs of theinvention can be single or double stranded.

As indicated above, the invention includes encoding sequences (DNA andRNA) and sequences complementary thereto. Such complementary sequencesinclude those that hybridize to a nucleic acid sequence encoding thesequence of SEQ ID NO:8, or variant thereof, or fragment thereof, underconditions of moderate or high stringency. As used herein, conditions ofmoderate stringency can be readily determined by those having ordinaryskill in the art based on, for example, the length of the DNA.Conditions are set forth by Sambrook et al, Molecular Cloning: ALaboratory Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring HarborLaboratory Press, (1989), and include use of a prewashing solution forthe nitrocellulose filters 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0),hybridization conditions of about 50% formamide, 6×SSC at about 42° C.,and washing conditions of about 60° C., 0.5×SSC, 0.1% SDS. Conditions ofhigh stringency can also be readily determined by the skilled artisanbased on, for example, the length of the DNA. Generally, such conditionsare defined as hybridization conditions as above, and with washing atapproximately 68° C., 0.2×SSC, 0.1% SDS. The artisan will recognize thatthe temperature and wash solution salt concentration can be adjusted asnecessary according to factors such as the length of the probe.

The invention also includes nucleic acids comprising sequences that areat least 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, or at least 99.9%identical to the sequence of SEQ ID Nos:1-4. The percent identity can bedetermined by visual inspection and mathematical calculation.Alternatively, the percent identity of two nucleic acids can bedetermined by comparing sequence information using BLAST 2 SEQUENCES.

The nucleic acids can bear a detactable label and/or can be bound to asolid support.

The present invention also relates to a recombinant molecule comprisinga nucleic acid as described above and to a host cell transformedtherewith. Using standard methodologies, well known in the art, arecombinant molecule comprising a vector and a nucleic acid encoding apolypeptide of the invention can be constructed. Vectors suitable foruse in the present invention include plasmid and viral vectors (e.g.,advenoviral, adeno-associated or retroviral vectors). Vectors into whicha nucleic acid can be cloned include any vectors compatible withtransformation into a selected host cell. The nucleic acids of theinvention can be present in the vector operably linked to regulatoryelements, for example, a promoter. Suitable promoters include, but arenot limited to the telomerase promoter, tumor specific promoters (e.g.,ovarian cancer—MISIIR promoter, colorectal cancer—CEA promoter, prostatecancer—PSA promoter).

As indicated above, the recombinant molecule of the invention can beconstructed so as to be suitable for transforming a host cell. Suitablehost cells include prokaryotic cells (e.g., bacterial cells) and lower(e.g., yeast) and higher eucaryotic cells (e.g., mammalian cells, suchas human cells). The recombinant molecule of the invention can beintroduced into appropriate host cells by one skilled in the art using avariety of known methods.

The present invention further relates to a method of producing apolypeptide of the invention. In one aspect, the method comprisesculturing the above-described transformed host cells under conditionssuch that the encoding sequence is expressed and the protein therebyproduced.

The functional potency of Letal to stimulate effector immune cells andincrease NKG2D expression makes possible new therapeutic strategies.Letal, and functional variants and fragments thereof, can be used, forexample, to enhance proliferation of immunoeffector cells (e.g., NKcells and NKT cells) and/or CTL activity both in vitro and in vivo andthereby modulate an immune response, for example, against tumors andinfectious agents (e.g., viruses and bacteria). In a preferredembodiment, Letal, or functional variants or fragments thereof, can beused to expand in vitro reactive T-cells or other effector cells, suchas TALL cells, for use in adoptive immunotherapy for patients withcancer and infectious (e.g., viral) diseases. In one approach to suchtherapies, artificial antigen presenting cells expressing Letal andcoated with anti-CD3 antibodies or specific tumor antigens can be used(see Maus et al, Nature Biotechnology 20:143 (2002)) in order toovercome the difficulty in obtaining sufficient numbers of CTLs. In thecase of cancer immunotherapy, tumor cells and tumor infiltratinglymphocytes can be isolated from a patient. Following transduction ofthe tumor cells with a Letal (or Letal variant or fragment) encodingsequence, the transduced cells can be incubated with the isolated Tcells to expand the population of T cells recognizing tumor antigen. Theresulting expanded population of tumor specific T cells can then beadministered (e.g., i.v.) to the patient to promote NK cytotoxicity andprovide costimulatory signals to CTL through NKG2D interactions. (Asregards details of these types of therapeutic approaches, see generallyLiebowitz et al, Curr Opin Oncol. 10(6):533-41 (1998) and U.S. Appln.No. 20020187151)).

It will be appreciated from a reading of this disclosure that, in thetreatment of autoimmune diseases (including rheumatoid arthritis), andin controlling rejection in patients undergoing tissue or organtransplantation, inhibition of expression or function of Letal can beuseful. In this regard, siRNA technology, for example, can be used toblock Letal expression and blocking agents (e.g., agents that blockbinding of Letal to the receptor) can be used to inhibit Letal function(e.g., blocking antibodies).

In addition to the foregoing, it will be apparent to one skilled in theart that Letal can serve as a marker for at least certain cancers. Byassaying for the presence of Letal (e.g., using anti-Letal antibodies),for example, in a tissue sample, the presence of at least certain tumorscan be detected. Letal detection can also be used as a means to monitorresidual or recurrent disease after treatment. Methods of using markerssuch as Letal for cancer detection are well known in the art.

Similarly, it will be appreciated that the nucleic acid sequences of theinvention can be used as probes and primers in detecting the presenceLetal genes or gene transcripts. Such detection can be useful, forexample, in cancer diagnosis.

In a further embodiment, the present invention relates to a previouslyunidentified molecule having the same C-type lectin structure as theNKG2D receptor. The encoding sequence for this molecule, designatedherein as LCCR, maps at chromosome 12, in the same cluster as the NKG2Dreceptor. As the NKG2D receptor, which is present on most CD8+ T-cells,γδ T-cells and NK cells, induces cytotoxicity by interacting withligands on the surface of tumor cells and cells infected by virus, it isexpected that LCCR also interacts with tumor cell ligands and/or ligandson the surface of virus infected cells and induces cytotoxicity againstthem.

A cDNA sequence encoding LCCR is as shown in FIG. 11 (SEQ ID NO:9) andthe predicted protein is shown in FIG. 12 (SEQ ID NO:10).

The invention includes the specific polypeptide shown in SEQ ID NO:10and variants and fragments thereof, as well as analogs and derivativesof such sequences.

LCCR variants include polypeptides substantially identical to thesequence of SEQ ID NO:10. The variants can include one or moredeletions, insertions, or substitutions relative to the sequence of SEQID NO:10 (e.g., substitutions of one or more of the amino acids of SEQID NO:10 wherein the substitution is with a conserved or non-conservedamino acid). The variant can have an amino acid sequence that is, forexample, at least, 50%, 60% or 70% identical to the sequence of SEQ IDNO:10, at least 80% identical, least 90% identical, at least 95%identical, at least 98% identical, at least 99% identical, or at least99.9% identical to the sequence of SEQ ID NO:10. The percent identitycan be determined, for example, by comparing sequence information usingBLAST 2 SEQUENCES. Variants in which differences in amino acid sequencerelative to the sequence of SEQ ID NO:10 are attributable to geneticpolymorphism (allelic variation among individuals producing the protein)are within the scope of the invention.

In certain aspects, this embodiment of the invention may not include thesequence corresponding to GenBank accession no. AF247788.

Fragments of this embodiment of the invention include, but are notlimited to, peptides/polypeptides comprising the cytoplasmic domain(e.g., about amino acid 1 to about amino acid 58 of the sequence of SEQID NO:10), the transmembrane domain (e.g., about amino acid 59 to aboutamino acid 81 of the sequence of SEQ ID NO:10), or the extracellulardomain (e.g., about amino acid 82 to about amino acid 231 of thesequence of SEQ ID NO:10), of the LCCR polypeptide of SEQ ID NO:10, orvariants thereof. The invention also includes fragments of thepolypeptide of SEQ ID NO:10, preferably, fragments comprising at least 5consecutive amino acids, more preferably, at least 10 or at least 20consecutive amino acids of the sequence of SEQ ID NO:10, or variantthereof. It will be appreciated that fragments of the invention can beemployed as immunogens, in generating antibodies.

Variants and fragments of the invention include, but are not limited to,polypeptides that retain a biological activity of the LCCR polypeptide,for example, the ability to bind ligands on tumor and/or virallyinfected cells and induce cytotoxicity against them. Polypeptides of theinvention can be tested for the ability to bind the such ligands in anysuitable assay, such as a conventional binding assay.

The invention also includes derivatives/analogs Of the LCCR polypeptideof SEQ ID NO:10 and variants and fragments thereof. The polypeptides ofthis embodiment of the invention can also be present as a fusionprotein, for example, to facilitate detection or isolation. Thepolypeptides can bear a detectable label and/or can be bound to a solidsupport.

The invention includes isolated and purified, or homogeneous,polypeptides of this embodiment of the invention, both recombinant andnon-recombinant. The polypeptides can be synthesized chemically usingart recognized techniques. The polypeptides of this embodiment of theinvention can be used as described below or can be used in theproduction of antibodies (polyclonal or monoclonal) using standardtechniques. The invention includes such antibodies, and binding portionsthereof, as well as their use, for example, in detecting the presence ofa polypeptide of this embodiment of the invention in a sample (in whichcase, the antibody can bear a detectable label).

The invention further relates to nucleic acid sequences (DNA or RNA)encoding the sequence of SEQ ID NO:10, or variants, and fragmentsthereof, or the complements of such encoding sequences. One specificnucleic acid sequence encoding the amino acid sequence of SEQ ID NO:10is set forth in SEQ ID No:9 (see also FIGS. 11 and 12). DNAs of theinvention can be single or double stranded. The nucleic acids can bear adetectable label and/or can be bound to a solid support.

As indicated above, this embodiment of the invention includes encodingsequences (DNA and RNA) and sequences complementary thereto. Suchcomplementary sequences include those that hybridize to a nucleic acidsequence encoding the sequence of SEQ ID NO:10, or variant thereof, orfragment thereof, under conditions of moderate or high stringency (asdefined above).

This embodiment of the invention also includes nucleic acids comprisingsequences that are at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, or atleast 99.9% identical to the sequence of SEQ ID NO:9. The percentidentity can be determined by visual inspection and mathematicalcalculation. Alternatively, the percent identity of two nucleic acidscan be determined by comparing sequence information using BLAST 2SEQUENCES.

The present invention also relates to a recombinant molecule comprisinga nucleic acid of this embodiment of the invention, as described above,and to a host cell transformed therewith. Using standard methodologies,well known in the art, a recombinant molecule comprising a vector and anucleic acid encoding a polypeptide of the invention can be constructed.Vectors suitable for use in the present invention include plasmid andviral vectors. Vectors into which a nucleic acid can be cloned includeany vectors compatible with transformation into a selected host cell.Such vectors include adenoviral, adeno-associated, retroviral andlentiviral vectors. The nucleic acids of this embodiment of theinvention can be present in the vector operably linked to regulatoryelements, for example, a promoter.

As indicated above, the recombinant molecule of this embodiment of theinvention can be constructed so as to be suitable for transforming ahost cell. Suitable host cells include prokaryotic cells and lower andhigher eucaryotic cells, such as mammalian cells, such as human cells.The recombinant molecule can be introduced into appropriate host cellsby one skilled in the art using a variety of known methods.

The invention further relates to a method of producing a polypeptide ofthis embodiment. In one aspect, the method comprises culturing theabove-described transformed host cells under conditions such that theencoding sequence is expressed and the protein thereby produced.

The identification of LCCR makes possible new therapeutic strategies,for example, immunotherapeutic approaches suitable for use in treatingtumors and viral infections, based on the induction of a cytotoxiceffect on the immune cells (e.g., NK cells) expressing LCCR. Suchstrategies can involve the use, for example, of gene therapy or DNAvaccination. Alternatively, soluble forms of the receptor (e.g., theextracellular domain) can be used to abrogate the action of stimulatoryligands in the case of autoimmunne disease treatment.

The invention includes compositions comprising the polypeptides of bothembodiments of the invention, nucleic acids, and/or antibodies asdescribed above and a carrier, diluent or excipient, e.g., apharmaceutically acceptable carrier diluent or excipient. Further, theinvention includes kits comprising such polypeptides, nucleic acidsand/or antibodies disposed within one or more container means.

Certain aspects of the invention are described in greater detail in thenon-limiting Examples that follow (see also Conejo-Garcia et al, CancerBiology and Therapy 2(4):e112-e117 (2003)). Attention is also directedto U.S. Pat. No. 6,458,350, US Appln. No. 20030147847 and to US Appln.No. 20020187151, the latter describing methods of treating neoplasiathat comprise administering ligands for the NKG2D receptor that can bepracticed using the ligand (or variant or fragment thereof) disclosedherein.

EXAMPLE 1

Experimental Details

Identification and characterization of the genomic and cDNA sequences ofLetal. The amino acid sequence of the α-1 and α-2 domains of all theknown human ligands for the NKG2D receptor (GenBank accession numbers:XM_(—)015542, XM_(—)027342, XM-015533, XM_(—)044229, and XM_(—)029639)were aligned in order to create patterns with the amino acids conservedin at least four of the five sequences and coded by a single exon.Genomic sequences at chromosome 6q25 were translated into the 6 possibleopen reading frames by using the ORF Finder Program(http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and were scanned for thepresence of these patterns with the PattinProt software(http://pbil.ibcp.fr/) (Combet et al, Trends Biochem. Sci. 25(3):147-150(2000)). Based on these criteria, a sequence was focussed upon that wasdesignated Letal. To search for 5′- and 3′-sequences of the novel gene,the amino acid sequences of ULBP1, ULBP2, and ULBP3 were used to performa scan algorithm for the detection of genes by using FGENESH+(http://genomic.sanger.ac.uk/gf/gfs.shtml) (Solovyev et al, NucleicAcids Res. 27(1):248-250 (1999)) and GeneBuilder(http://125.itba.mi.cnr.it/˜webgene/genebuilder.html) (Milanesi et al,15(7-8):612-621 (1999)). The primers Letal.F:5′-CCATACCAGTGAGGGTGAATG-3′ (SEQ ID NO:11) and Letal.R:5-CCCATGATTCACCTCTCTTGAG-3′ (SEQ ID NO:12) were used to amplify by PCRthe complete open reading frame for the predicted gene from the ovariancarcinoma cell line A2008. The putative cleavage sites of theprepropeptide were predicted with SignalP V2.0(http://www.cbs.dtu.dk/services/SignalP-2.0) (Nielsen et al, ProteinEng. 10(1):1-6 (1997)) and the transmembrane domain with PRED-TMRsoftware (http://o2.db.uoa.gr/PRED-TMR/) (Pasquier et al, Protein Eng.12(5):381-386 (1999)). Clustalw (http://www.ebi.ac.uk/clustalw) was usedto perform alignments and build the phylogenetic tree and PredictProtein(http://maple.bioc.columbia.edu/pp/) (Rost and Sander, J. Mol. Biol.232(2):584-599 (1993)) was used to predict the secondary structure.

Quantification of Letal by real-time quantitative RT-PCR. Letalexpression was analyzed by TaqMan analysis as previously described(Garcia et al, FASEB J 15(10):1819-1821 (2001)). The Letal systemconsisted of the primers: Letal.F, 5′-CTCAGGATGCTCCTTTGTGACAT-3′ (SEQ IDNO:13); Letal.R, 5′-CTTCACGTTGACAAAACATCTCG-3′ (SEQ ID NO:14), and theprobe Letal.P, 5′-(FAM)CCCAGATAAAGACCAGTGATCCTTCCACT (SEQ ID NO:15)(TAMRA)-3′. The cDNA load was normalized to human GAPDH with primersGAPDH F: 5′-CCTGCACCACCAACTGCTTA-3′ (SEQ ID NO:16) and GAPDH R:5′CATGAGTCCTTCCACGATACCA-3′ (SEQ ID NO:17) and the probe GAPDH.P:5′(FAM) CCTGGCCAAGGTCATCCATGACAAC (TAMRA)-3′ (SEQ ID NO:18).

Tissues, cell lines and purification of cells. Normal human tissues wereobtained from the Cooperative Human Tissue Network (Zhang et al, N.Engl. J. Med. 348(3):203-213 (2003)). A2008 ovarian carcinoma cell linewas treated for 24 h with serum-free medium (control) or serum-freemedium containing either 10 U/ml interleukin (IL)-1β, 10 ng/ml tumornecrosis factor (TNF-α), 40 ng/ml interferon (IFNγ), 0.5 μg/mlLypopolyssaccaride (LPS), or 10 ng/ml TNF-α plus 40 ng/ml IFN-γ. 10ng/ml PMA was kept for 4 hs and retinoic acid for 24, 48, and 72 hs. Allthe cytokines were from Peprotech (Rocky Hill, N.J.), except retinoicacid (Sigma, St Louis, Mo.). Additionally, cells were cultured in mediawithout glucose for 48 hs or under hypoxia (1.5% O₂) for 16 hs. Freshperipheral blood lymphocytes were obtained by leukapheresis andelutriation. CD8⁺ cells were prepared by negative selection using theOKT4 antibody (Maus et al, Nat. Biotechnol. 20(2):143-148 (2002)).

Constructs and generation of antibodies. SKOV3 and K562 cells weretransduced using retroviral vector MIGR1, generously provided by WarrenPear (University of Pennsylvania). Letal or mock transductantsexpressing equivalent levels of the green fluorescent protein weresorted and cultured by standard procedures. To demonstrate the bindingof Letal to the NKG2D immnoreceptor, Letal⁺/mock-transduced SKOV3 andK562 cells were saturated with 10% mouse serum, and incubated afterwashing with 3 μg/ml of recombinant human NKG2D/Fc chimera (R & DSystems, Minneapolis, Minn.). Anti-human IgG mAb (G18-145; Pharmingen,San Diego, Calif.) was used to detect the chimerical molecule.

To generate a polyclonal anti-Letal Ab, C57BL/6 mice were immunized at0, 1 and 2 weeks with 25 μg of Letal cDNA cloned in the pcDNA 3.1expression vector (Invitrogen). Positive sera were subsequentelyconfirmed by flow cytometry using different cell lines transfected withthe complete Letal Open Reading Frame.

GPI-specific phospholipase-C treatment, ⁵¹Cr release and apoptosisassay. Letal⁺ and Letal⁻ A2780 cells were treated with 2 U/mlGPI-specific phospholipase-C (Sigma) at 37° C. for 1 hr. A standard⁵¹Cr-release assay was performed using 8,000 ⁵¹Cr-labeled targets/well.

CD8⁺ lymphocyte stimulation and cytokine release. To analyze the effectsof Letal on the T-cell proliferation and production of cytokines, apreviously described artificial antigen-presenting cell (aAPC) model wasused (Maus et al, Nat. Biotechnol. 20(2):143-148 (2002)). Briefly, thesystem is based on the stable expression of the human low-affinity Fcγreceptor, CD32, on K562 cells (K32 cell line). K32 cell-based aAPCs wereadditionally transduced with Letal or with the empty vector as describedabove, irradiated with 100Gy, and washed twice with RPMI medium.Letal⁺-K32 cells or mock transductants were loaded, when indicated, withanti-CD3 (OKT3), or anti-CD3 plus anti-CD28 (mAbs; 9.3) monoclonalantibodies at 0.5 μg/ml for 10 min at room temperature. Loaded &APCswere mixed with CD8⁺ T-cells at a 1:2 ratio and the T-cell concentrationwas maintained at 0.5×10⁶ cells/ml throughout the culture. Cultures werepulsed with 1 μCi of [³H] thymidine from day 2 to day 5 and incorporatedradioactivity was determined using a 1450 Microbeta scintillationcounter (Wallac, Turku, Finland). The amounts of secreted IL-2 and IFNγwere determined by commercial ELISA, following the manufacturer'sinstructions (R & D Systems). Flow cytometry was performed with aFACScalibur (BD Biosciences, San Jose, Calif.). Mouse anti-humanmonoclonal antibody 149810 (R & D Systems) was used to evaluate theexpression of NKG2D.

Results

Analysis of the genomic and cDNA sequences of Letal. The cloneNT_(—)007295.3 from the Human Genome Project, containing the sequencesof all the ULBPs mapping at chromosome 6q25, was translated into the sixpossible open reading frames and they were screened for the presence ofthe pattern “L-Q-X(4)-C-E-X(7)-R-G-S-X(2)-F-X(3)-G-X(2)-F-L-X(6)-W-T”.Based on these criteria, a sequence that was named Lymphocyte Effectorcell Toxicity-Activating Ligand (Letal) was focussed upon. Differentparts of the cDNA for Letal were bioinformatically deduced and itsfull-length was amplified from the ovarian carcinoma cell line A2008(GenBank Accession number: AY069961; submitted on Dec. 12^(th), 2001).The gene exhibited a 792-bp open reading frame encoding a protein of 263amino acids in length (FIG. 1A). Letal was found to be identical to agenomic fragment identified as RAET1E, potentially encoding the first222 amino acids (Radosavljevic et al, Genomics 79(1):114-123 (2002)).Sequence identity of Letal with ULBP1, ULBP2, and ULBP3 ranged from 33.3to 38.5% (FIG. 1B). As shown in FIG. 1C, molecular phylogenetic analysisconfirmed the relatedness of Letal to human ULBPs and to the recentlydescribed murine MULT1 (Carayannopoulos et al, J. Immunol.169(8):4079-4083 (2002)). As with ULBPs, the corresponding proteincomprised a class I MHC-like α-1α-2 platform domain. However, it did notencompass glycosylphosphatidyl inositol (GPI) transamidation sites andinstead exhibited, uniquely within this group of NKG2D ligands, acytoplasmic domain.

Letal is a ligand of NKG2D expressed in a variety of normal tissues. Todemonstrate that Letal is a ligand for NKG2D, a retroviral system wasused to express Letal in erythroleukemia MHC-I^(neg) K562 cells.Expression of the Letal protein was confirmed on transduced cells, butnot on mocked transductants, by flow cytometry using serum ofLetal-immunized mice (FIG. 1D). To confirm the prediction that Letaldoes not contain GPI transamidation sites, Letal⁺ K562 cells weretreated with GPI-specific phospholipase-C. Instead of decreasing thebinding of anti-Letal Ab, enzymatic treatment resulted in strongerstaining in three different experiments, suggesting the cleavage ofmolecules that interfere with the exposure of Letal epitopes, andconfirming the predicted transmembrane structure of Letal. To evaluatethe binding of NKG2D to transduced Letal, a recombinant soluble NKG2D-Fcfusion protein was used, containing the ectodomain of the immunoreceptor(Phe78 through Val216). Using a monoclonal antibody against human IgG,markedly stronger staining was observed in Letal⁺ K562 and Letal⁺ SKOV3cells than in mock-transductants by flow cytometry (FIG. 1D), confirmingthat Letal is a ligand for NKG2D. Treatment with phospholipase Cdecreased binding of sNKG2D to Letal⁺ K562 cells, although binding washigher than to control cells. No significant decrease was observed withLetal⁺ SKOV3 cells. These results indicate that cleavage of otherGPI-anchored NKG2D ligands, but not Letal, accounted for the differencein sNKG2D binding observed between treated and control Letal⁺ K562cells.

Using real-time quantitative PCR (TaqMan), abundant Letal transcriptswere detected in normal small intestine by, and at lower levels innormal brain, breast, colon, spleen, skeletal muscle, uterus, thymus,placenta, blood lymphocytes and ovary (FIG. 2A). RT-PCR analysisindicated the presence of Letal mRNA in most colon cancer cell linestested, but only in two out of fifteen ovarian carcinoma cell lines(FIG. 2B). A shorter splicing variant encoding for a protein lacking 36amino acids from the α-1 domain, corresponding to the GenBank Entry:AY054974, was found in 4 cancer cell lines. However, Letal wasinvariably the predominant form. No expression in immature dendriticcells was detected. Basal Letal mRNA levels increased 1.6-fold and3-fold in A2008 cells upon infection with Herpes simplex virus oraddition of TNF-α, respectively, whereas other inflammatory cytokines,hypoxia or starvation had little to no effect on Letal expression (FIG.3A). Human MICA/B and mouse RAE-1 family members are upregulated by48-72 hr treatment with retinoic acid (RA) (Cerwenka et al, Immunity12(6):721-727 (2000), Jinushi et al, Int. J. Cancer 104(3):354-361(2003)). Surprisingly, a progressive decrease of Letal mRNA expressionwas found after treatment of A2008 cells with RA, which was maximum(6-fold) after 72 hr stimulation, suggesting that signals inducingtranscriptional activation of NKG2D ligands are markedly different foreach molecule (FIG. 3B).

Sustained Letal engagement increases the expression of NKG2D in CD8⁺lymphocytes. It has been reported that MICA engagement for up to 48 hrcauses downregulation of NKG2D and, in turn, impairment of T-cellactivation (Groh et al, Nature 419(6908):734-738 (2002)). To determinewhether Letal influences NKG2D expression, K32 artificialantigen-presenting cells (aAPCs) (K562 cells transfected with humanCD32) were transduced (Maus et al, Nat. Biotech. 20:143 (2002)) withLetal⁺ or control retrovirus, irradiated and cultured with peripheralCD8⁺ cells. CD8⁺ T-cells derived from peripheral blood showed a low meanfluorescence intensity staining of NKG2D after co-culture withmock-transduced K32 erythroleukemia cells for 4 days (FIG. 2C). Insteadof decreasing the expression of NKG2D, incubation of lymphocytes withLetal⁺ K32 cells resulted in a slight increase (2-fold), suggesting thateither a transcriptional mechanism compensates the initial degradationof NKG2D, or that the effects of Letal are different from that of MICA.This increase was more evident after loading Letal⁺ K32 cells withanti-CD3 mAb (3-fold), whereas CD3/CD28 signaling resulted in thehighest up-regulation (4.5-fold increase). Culture of CD8⁺ T-cells withLetal⁻ K32 cells loaded with anti-CD3 mAb produced the same result thanCD3/Letal stimulation.

Letal induces T-cell receptor-dependent proliferation and Tc1polarization in CD8⁺ lymphocytes. To determine whether Letal influenceslymphocyte activation, control or Letal⁺ K32 aAPCs were loaded withanti-CD3 and/or anti-CD28 mAb, and incubated for 2 to 5 days withperipheral CD8⁺ cells. Proliferation was similar in the presence of CD3mAb/Letal and CD3/CD28 mAbs (FIG. 3A). Anti-CD3/Letal stimulationincreased day-2 production of IL-2 27-fold and day-3 IFN-γ secretion83-fold compared to CD3 stimulation alone (FIGS. 3B and 3C). Notably,CD8⁺ cell expansion was markedly lower with CD3 stimulation in theabsence of Letal in three independent experiments (29% fewer CD8⁺ cellsat day 5). No activation in the absence of anti-CD3 mAb was observed.Taken together, these data demonstrate that Letal is a potentcostimulatory molecule for the αβ T-cell receptor, enhancing CD8⁺ cellproliferation and inducing Tc1 responses.

Expression of Letal induces the killing of cancer cells by CD8⁺ and NKcells. Letal-induced cytotoxic effects were next analyzed in aredirected lysis experiment of K562 cells. As expected,CD3/Letal-activated CD8⁺ cells effectively killed MHC-I^(neg) K562erythroleukemia cells bearing anti-CD3 antibody. In the absence of a TCRsignal, Letal engagement alone was markedly less effective (FIG. 4A),while Letal alone-activated lymphocytes failed to kill K562 cells.

For analysis of the NK-dependent anti-tumor immune response, thecytotoxicity of NK cells against Letal⁺ or control SKOV3 chemoresistantovarian carcinoma cells was compared. Letal expression increased killingof SKOV3 cells by IL-15 activated NK effectors (FIG. 4B), whileuntreated NK cells could not kill tumor cells efficiently.

Summarizing, the foregoing study resulted in the characterization of thefirst human transmembrane NKG2D ligand lacking a α-3 domain (Letal).Letal is expressed by tumors and acts as a costimulatory ligandpromoting CTL activation, expansion, type-1 polarization andcytotoxicity. Moreover, Letal is directly involved in the activation ofNK cell-mediated anti-tumor cytotoxicity. Letal maps to chromosome 6q25,and is identical to a suggested partial sequence lacking 41 amino acids,found through a previous analysis of genomic sequences around the ULBPcluster (Radosavljevic et al, Genomics 79(1):114-123 (2002)). Like theULBPs, the corresponding Letal protein contains a class I MHC-likeα-1α-2 platform domain. However, Letal differs by exhibitingtransmembrane and cytoplasmic domains. The highest sequence identitybetween Letal and a ULBP protein is 38.5%. Interestingly, a splicingvariant of Letal has been found in most colon cancer cell linesevaluated. The corresponding peptide lacks 36 amino acids from the α-1domain and corresponds to an unpublished GenBank entry named as retinoicacid early inducible (RAE)-1-like transcript 4. To the contrary, it hasbeen demonstrated that, instead of being up-regulated by retinoic acid,Letal is down-regulated. This, taken together with the low sequenceidentity between Letal and RAE-1 family members, suggests that the nameRAE may be inexact. Given the functional potency of Letal to stimulateeffector immune cells and increase NKG2D expression, retinoicacid-dependent mechanisms could be used to control T-cell proliferationand prevent widespread inflammation through Letal down-regulation.Interestingly, in the human, MICA/B are also up-regulated by retinoicacid (Jinushi et al, Int. J. Cancer 104(3):354-361 (2003)). Thisapparent contradiction may be explained by a differential expression ofactivating molecules in different cell types. Alternatively, NKG2D maybind different ligands with different affinities. Moreover, little isknown about the expression of these proteins in vivo. It is possiblethat MICA/B up-regulated by retinoic acid are enzymatically cleaved,thus releasing soluble forms that down-regulate NKG2D expression (Grohet al, Nature 419(6908):734-738 (2002)), finally producing the samediminishing effects.

EXAMPLE 2

Experimental Details

Quantification of Letal by real-time quantitative RT-PCR. Letalexpression was analyzed by TaqMan analysis as previously described(Garcia et al, FASEB J. 15:1819-1821 (2001)). The Letal system consistedof the primers: Letal.F, 5′-CTCAGGATGCTCCTTTGTGACAT-3′ (SEQ ID NO:13);Letal.R, 5′-CTTCACGTTGACAAAACATCTCG-3′ (SEQ ID NO:14), and the probeLetal.P, 5′-(FAM)CCCAGATAAAGACCAGTGATCCTTCCACT (TAMRA)-3′ (SEQ IDNO:15). The cDNA load to human GAPDH was normalized with primers GAPDHF: 5′-CCTGCACCACCAACTGCTTA-3′ (SEQ ID NO:16) and GAPDH R:5′-CATGAGTCCTTCCACGATACCA-3′ (SEQ ID NO:17) and the probe GAPDH.P:5′(FAM) CCTGGCCAAGGTCATCCATGACAAC (SEQ ID NO:18) (TAMRA)-3′. Theexpression of phospholipase-A2 (GLPD1) was quantified using the SYBRGreen Master Mix kit (Applied Biosystems) and primers Ph.F:5′-GCAATGATGTACTGTCTCTTTTGGA-3′ (SEQ ID NO:19) and Ph.R:5′-CAACCTCAGCCAAGTAACGGTAG-3′ (SEQ ID NO:20).

Tissues, cell lines and purification of cells. Normal human tissues wereobtained from the Cooperative Human Tissue Network. Ovarian tumorspecimens were obtained from the University of Turin, Italy (Zhang etal, N. Engl. J. Med. 348:203-213 (2003)). For the analysis oftumor-infiltrating lymphocytes, ovarian tumors were minced and digestedwith collagenase A (Roche, Mannheim, Germany), and cell sorting wasperformed on a MoFlo cell sorter (Cytomation, Fort Collins, Colo.) witha proportion of the filtered suspension. Fresh peripheral bloodlymphocytes were obtained by leukapheresis and elutriation. CD8⁺ cellswere prepared by negative selection using the OKT4 antibody (Maus et al,Nat. Biotechnol. 20:143-148 (2002)).

Constructs and generation of antibodies. K562 cells were transducedusing retroviral vector MIGR1, generously provided by Warren Pear(University of Pennsylvania). Letal or mock transductants expressingequivalent levels of the green fluorescent protein were sorted andcultured by standard procedures.

To generate a polyclonal anti-Letal Ab, C57BL6 mice were immunized at 0,1 and 2 weeks with 25 μg of Letal cDNA cloned in the pcDNA 3.1expression vector (Invitrogen). Positive sera were subsequentelyconfirmed by flow cytometry using different cell lines transfected withthe complete Letal Open Reading Frame.

Apoptosis assay. The percentage of apoptotic cells was determined after17 h incubation with 50 μM cisplatin or 18 hs exposure to 0.1 μg/mlanti-CD95 mAb (EOS9.1; Pharmingen) by using the TACS annexin-V apoptosisdetection kit, according to the manufacturer's instructions (R & DSystems). Fas expression was determined by flow cytometry by usingEOS9.1 as a primary Ab and a PE-labeled anti-mouse IgM (R6-60.2;Pharmingen) as a second Ab.

CD8⁺ lymphocyte stimulation, cytokine release and glucose metabolismanalysis. To analyze the effects of Letal on the T-cell proliferationand production of cytokines, a previously described artificialantigen-presenting cell (aAPC) model was used (Maus et al, Nat.Biotechnol. 20:143-148 (2002)). Briefly, the system is based on thestable expression of the human low-affinity Fcγ receptor, CD32, on K562cells (K32 cell line). K32 cell-based aAPCs were additionally transducedwith Letal or with the empty vector as described above, irradiated with100Gy, and washed twice with RPMI medium. Letal⁺-K32 cells or mocktransductants were loaded, when indicated, with anti-CD3 (OKT3), oranti-CD3 plus anti-CD28 (mAbs; 9.3) monoclonal antibodies at 0.5 μg/mlfor 10 min at room temperature. Loaded aAPCs were mixed with CD8⁺T-cells at a 1:2 ratio and the T-cell concentration was maintained at0.5×10⁶ cells/ml throughout the culture. Cultures were pulsed with 1 μCiof [³H] thymidine from day 2 to day 5 and incorporated radioactivity wasdetermined using a 1450 Microbeta scintillation counter (Wallac, Turku,Finland). The amounts of secreted IL-2 and IFN-γ were determined bycommercial ELISA, following the manufacturer's instructions (R & DSystems). Flow cytometry was performed with a FACScalibur (BDBiosciences, San Jose, Calif.). Mouse anti-human monoclonal antibody149810 (R & D Systems) was used to evaluate the expression of NKG2D.Glut-1 intracellular staining and glucose uptake were performed exactlyas previously described (Frauwirth et al, Immunity 16:769-777 (2002)).

Laser capture microdissection and immunohistochemistry.Hematoxylin-labeled tumor islands were microdissected from six μm thickcryosections using the μCUT Laser-MicroBeam System (SL Microtest, Jena,Germany), according to the manufacturer's instructions. RNA wasimmediately extracted from captured tissue by using the PicoPure RNAIsolation kit (Arcturus, Mountain View, Calif.). Immunohistochemistrywas performed exactly as previously described (Zhang et al, N. Engl. J.Med. 348:203-213 (2003)).

Results

CD8⁺ T-cells represent the predominant NKG2D⁺ population in advancedovarian carcinomas. The presence of total leukocytes in 100 snap-frozenspecimens of ovarian carcinomas was first evaluated byimmunohistochemistry. CD45⁺ leukocytes were detected in differentproportions within tumor-cell islets, in stroma, or both. CD45⁺ cellsrepresented up to 25% of total cells in selected specimens (FIG. 6A).Next, the expression of NKG2D by tumor-infiltrating leukocytes wasexamined. More than 50% of tumor islets in these specimens wereinfiltrated by NKG2D⁺ cells, which, in average, represented 15% of thetotal leukocytes in stage III tumors (FIG. 6B). The relativecontribution of different effector cell types, i.e. CD8⁺ and NK cells,in these tumors was investigated. The vast majority of effector cellswere found to be CD8⁺ T-cells (FIG. 6C), while NK cells were scarcelyrepresented in most specimens analyzed (FIG. 6D), suggesting apredominant role of T-cell mediated responses in immunosurveillanceagainst established tumors. Interestingly, lower stage tumors containedsignificantly fewer infiltrating lymphocytes than advanced ovariancarcinomas, suggesting that a certain degree of invasion anddedifferentiation is necessary to trigger a sustained immune response.

CD8⁺ T-cells infiltration is associated with Letal overexpression inhuman advanced ovarian carcinomas. Since ovarian cancer progression isassociated with an increasing number of infiltrating lymphocytes, Letalexpression during tumor progression was next analyzed in 48 ovarianneoplasms and control postmenopausal ovaries. Strong immunostaining forLetal was detected in tumor islets of stage III carcinomas (FIG. 6E,6F). A positive signal was also observed in select tumor-inflitratingleukocytes within peritumoral stroma. Using Real-Time PCR, it was foundthat expression of Letal mRNA was low in normal ovaries and benign orlow-malignant potential (borderline) tumors, whereas stage I to IIIovarian carcinomas exhibited markedly higher expression (higher LetalmRNA expression in malignant tumors (stage III and stage I; n=38) vsborderline and benign tumors, plus normal post-menopausal ovaries (n=10;p=0.02); higher Letal mRNA expression in stage III tumors (n=29) vsstage I, borderline and benign tumors, plus normal post-menopausalovaries (n=19; p<0.05); FIG. 7A). The average Letal mRNA levels in stageI carcinomas were 10-fold higher compared to non-malignant specimens,whereas Letal expression was 100-fold higher in stage III compared tostage I cancers.

T-cells infiltrate tumor islets (intratumoral T-cells) in approximately55% of ovarian cancers, while T-cells are exclusively detected inperitumoral stroma in the remainder (zhang et al, N. Engl. J. Med.348:203-213 (2003)). Simultaneous stimulation of the T-cell receptor andLetal induces proliferation of cytotoxic lymphocytes in vitro(Conejo-Garcia et al, Cancer Biol. Ther. 2 available online). Toinvestigate whether Letal plays any role in the expansion ofintratumoral T-cells in vivo, Letal levels in tumor islets showingintratumoral T cells and tumor islets lacking intratumoral T-cells weremeasured, using laser capture microdissection to procure highly puresamples of tumor islets. TaqMan analysis of 19 different stage IIIspecimens revealed a 30-fold higher Letal mRNA expression in isletsinfiltrated by T-cells compared to islets lacking T-cells (P=0.041; FIG.7B). Since most ovarian tumors express MHC-I by immunohistochemistry(Kooi et al, Cell Immunol. 174:116-128 (1996)) and flow cytometry, thesedata suggest that Letal may be involved in the enrichment of T-cells intumor islets.

Because, patients with ovarian cancer whose tumors exhibit T-cellsinfiltrating tumor islets (TILs) experience better outcome (Zhang et al,N. Engl. J. Med. 348:203-213 (2003)), the survival of patients withstage-III ovarian cancer was analyzed based on the expression of LetalmRNA. There were significant differences in the distribution of overallsurvival (log-rank test; P=0.015; FIG. 7C). Patients whose tumorsexpressed Letal mRNA had a median overall survival of 37 months (n=29),as compared with 20 months among patients with Letal-negative tumors(n=9). The five-year overall survival rate was 41% among patients whosetumors expressed Letal mRNA but only 22% among patients whose tumorswere Letal-negative. Thus, although expression of Letal increases inlate stage, it appears to play a protective role in ovarian carcinoma.

It has been reported that engagement of soluble forms of NKG2D ligandscauses downregulation of NKG2D and, in turn, impairment of T-cellactivation (Groh et al, Nature 419:734-738 (2002)). To test whetherdifferent glycosylphosphatidylinositol (GPI)-anchored NKG2D ligands(Cosman et al, Immunity 14:123-133 (2001)) may be secreted by enzymaticcleavage in vivo and impair the expression of NKG2D, the expression ofthe GPI-specific phospholipase-D (GLPD1) was analyzed in the same tumorspecimens. As determined by TaqMan, GLPD1 mRNA levels were significantlyhigher in malignant (stage I or III) than in benign tumors (P=0.04; FIG.2D). This data suggests a possible mechanism of immune evasion which cancounteract of the immunostimulatory effect of Letal.

Tumor-infiltrating CD8⁺ lymphocytes do not express CD28 but can beexpanded through CD3/Letal engagement. A significant proportion ofperipheral effector CD8⁺ cells are known to be negative for thecostimulatory molecule CD28, thus antigen-induced proliferative responsemay be impaired, even after addition of exogenous IL-2, or relysubstantially on alternate costimulatory receptors (Azuma et al, J.Immunol. 150:147-1159 (1993)). The expression of CD28 on CD8⁺lymphocytes from three dissociated ovarian tumors and two tumor ascitesspecimens was analyzed. Diminished or completely absent expression ofCD28 was observed on greater than 80% of tumor-infiltrating cytotoxiclymphocytes, as well as in the vast majority of CD8⁺ lymphocytes fromtumor ascites (FIG. 8A). As tumor-infiltrating leukocytes express NKG2Dby immunohistochemistry (FIG. 6B). An investigation was made as towhether Letal engagement could compensate for the absence of CD28 inCD8⁺ CD28⁻ lymphocytes sorted from the same specimens. As shown in FIG.8B, CD3/Letal stimulation delivered through the K562 artificialantigen-presenting system induced a sustained proliferation of CD8⁺cells from all specimens for at least 3 weeks. Additionally, secretionof IFN-γ was dramatically increased with respect to activation byTCR-CD3 alone (FIG. 8C). Thus, Letal provides an importanttumor-associated costimulatory molecule recognized by tumor-associatedCD28⁻ CTL. The above findings collectively support an important role ofLetal in the intraumoral expansion of TILs in ovarian carcinoma.

Letal signaling increases glucose transporter expression and glucoseuptake during T-cell activation. It has been recently reported thatlymphocyte activation through CD28 costimulation increases glycolyticflux (Frauwirth et al, Immunity 16:769-777 (2002)). To test whetherLetal may have a similar effect during T-cell activation, peripheralCD8⁺ T-cells were stimulated for 20 hr with anti-CD3,anti-CD3/anti-CD28, anti-CD3/Letal, or Letal alone and the expression ofthe glucose transporter Glut-1 was analyzed by flow cytometry.Activation by cross-linking the TCR/CD3 complex altered Glut-1expression only in 11% of the cells (FIG. 9A). In contrast, stimulationwith anti-CD3/Letal or Letal alone led to a dramatic induction of Glut-1expression, which was similar to that induced by CD3/CD28 costimulation.

Glucose uptake rates were next measured with [³H]-2-deoxyglucose incells stimulated as described above. As expected, anti-CD3/anti-CD28increased glucose uptake to previously reported levels, while CD3 alonehad little to no effect. Interestingly, the uptake rate increase waseven more apparent in CD3/Letal and Letal alone-stimulated cells (FIG.9B). Thus, a signal transduction induced by Letal alone prepareslymphocytes for the increased metabolic demands associated with immuneresponses.

The studies in ovarian cancer indicated that the presence ofintratumoral T cells strongly predicts prolonged remission followingcytotoxic chemotherapy (Zhang et al, N. Engl. J. Med. 348:203-213(2003)). Cytotoxic chemotherapy can, however, deplete tumor-specificeffector T-cells (Lee et al, Nat. Med. 5:677-685 (1999)). Because theglycolytic pathway is implicated in lymphocyte survival (Plas et al,Nat. Immunol. 3:515-521 (2002)), a determination was made as to whetherLetal could protect T-cells from early apoptotic death induced bygenotoxic drugs. Incubation with 50 μM cisplatin for 17 hr produced ahigher percentage of apoptotic cells in lymphocytes pre-stimulated for 3days with anti-CD3 than in resting lymphocytes. In contrast, apoptosisof CD3/Letal and CD3/CD28 pre-stimulated CD8⁺ cells was markedly lower(FIG. 9C). Furthermore, T-cells pre-stimulated with Letal alone showedmarkedly stronger protection from platinum-induced apoptosis.Collectively, the above data demonstrate that Letal engagement protectsCD8⁺ T-cells from apoptosis induced by TCR-dependent mitogenic signalsand genotoxic drugs.

Letal engagement protects CD8⁺ T-cells from FasL-induced apoptosis.Ovarian carcinomas harbor abundant intratumoral T cells and the latterexhibit evidence of activation and are associated with dramaticallyimproved clinical outcome (Zhang et al, N. Engl. J. Med. 348:203-213(2003)). Because ovarian carcinomas express FasL and otherlymphocyte-depleting death ligands (Rabinowich et al, J. Clin. Invest.101:2579-2588 (1998)), the prevalence of FasL expression and its impacton TILs was determined in 42 stage III ovarian carcinoma specimens.Strong FasL immunoreactivity was detected in tumor cells in areas ofselected specimens (FIG. 10A), as well as in a proportion of stromalleukocytes. To test whether Letal promotes the generation of activatedT-cell subpopulations that are able to resist the pro-apoptotic tumormicroenvironment, peripheral CD8⁺ T-cells were stimulated for 4 dayswith CD3/Letal, CD3/CD28, or anti-CD3 alone, and expression of Fas wasmeasured by flow cytometry. As shown in FIG. 10B, Fas expression wasmarkedly higher in T-cells stimulated with anti-CD3 mAb compared toCD3/CD28 costimulated T-cells. These data agree with previous reports onTCR activation-induced apoptosis (Zaks et al, J. Immunol. 162:3273-3279(1999)). Fas expression in unstimulated lymphocytes progressivelyincreased, reaching similar levels to lymphocytes costimulated withCD3/CD28 on day-4. In contrast, Letal engagement dramatically reducedFas expression in 60% of T-cells compared to controls, suggesting thatLetal engagement protects CD8⁺ lymphocytes from FasL-induced apoptosis.CD8⁺ cells stimulated for 3 days with different ligands were thereforeexposed to anti-CD95 agonistic antibody EOS9.1 and (early) apoptosis wasquantified by flow cytometry analysis of annexin-V staining. Asexpected, the percentage of non-apoptotic cells amongCD3/Letal-stimulated T-cells was dramatically higher than among T-cellsstimulated with CD3/CD28, CD3 alone or control T-cells (FIG. 10C).Therefore, Letal confers CD8⁺ lymphocytes the ability to resistsuicidal, fratricidal, and tumor-induced apoptosis induced by tumordeath ligands.

Summarizing, the data show that human advanced ovarian carcinomaexhibiting improved outcome and stronger lymphocyte infiltration alsoshow higher levels of the NKG2D ligand Letal. Cytotoxic lymphocytessorted from these tumors were negative for CD28, but Letal exertedmarked costimulatory properties on TCR-mediated proliferation of thesecells. Moreover, Letal engagement protects CD8⁺ T-cells from apoptosisinduced by TCR-dependent mitogenic signals, tumor death ligands andgenotoxic drugs. Since NKG2D is an important activating receptor forCD8⁺ lymphocytes and NK cells in peripheral tissues, these results havemarked implications for tumor immunosurveillance. NK cells may beresponsible for rejection of tumors at early stages of malignanttransformation (Diefenbach et al, Nature 413:165-171 (2001), Cerwenka etal, Proc. Natl. Acad. Sci. USA 98:11521-11526 (2001)). However, CD8⁺cells are markedly more frequent than NK cells in advanced ovariancarcinoma. The presence of tumor infiltrating T-cells correlates withMHC class-I expression of tumor cells in ovarian cancer (Kooi et al,Cell Immunol. 174:116-128 (1996)). This suggests a predominant role forTCR-dependent immune response against established tumors. The presenceof T-cells infiltrating tumor islets is associated with dramaticallylonger survival and prolonged remission in ovarian carcinoma (Zhang etal, N. Engl. J. Med. 348:203-213 (2003)). Interestingly, significantlyhigher expression of Letal was found in tumor islets exhibitingaccumulation of CD3⁺ cells. Collectively, these findings suggest thatimmune surveillance against advanced ovarian carcinoma is mainlyaccomplished through expansion of tumor-specific CTL. Given the role ofLetal in promoting the survival, proliferation and cytotoxicity of CD8⁺cells, tumor-associated Letal may play an important role in promotingthe expansion of tumor-specific CTLs in the context of MHC-I expression.It is known that upon repeated stimulation with antigen, particularly inthe presence of IL-2, T-cells become susceptible to the induction ofFas-mediated apoptosis (Plas et al, Nat. Immunol. 3:515-521 (2002)). Ithas been proposed that increased resistance of T cells to apoptosis is anecessary condition for the establishment of chronic inflammatorydiseases and is required for the orchestration and endurance ofsustained immune responses (Levine et al, Semin. Immunool. 13:195-199(2001), Westermann et al, Ann. Intern. Med. 135:279-295 (2001)). Inlymph nodes, activation-induced T cell apoptosis is inhibited bycostimulatory signals provided by professional antigen-presenting cellsthrough CD28, CD7 or some members of the TNF receptor family. Engagementof CD28 involves activation of MAP kinases ERK, p38 and JNK; activationof NF-κB; upregulation of Bcl-2, Bcl-x_(L) and c-FLIP; anddownregulation of Fas (Kataoka et al, Curr. Biol. 10:640-648 (2000),Khoshnan et al, J. Imunol. 165:1743-1754 (2000), Kirchhoff et al, Eur.J. Immunol. 30:2765-2774 (2000)). In the periphery, NKG2D serves as oneof the most potent costimulatory receptors for CD8⁺ effectorlymphocytes. Engagement of NKG2D by Letal was found to markedly decreaseexpression of Fas and significantly reduced TCR activation-inducedapoptosis. The survival-promoting effect of Letal was not restricted tothe Fas pathway however, as Letal engagement protected lymphocytes alsofrom death induced by the genotoxic drug cisplatin. This finding hasimportant implications for the effects of chemotherapy on antitumorimmune response, as activated lymphocytes become susceptible to toxicmetabolites and cytotoxic chemotherapy has been shown to depletetumor-reactive T cells (Zaks et al, J. Immunol. 162:3273-3279 (1999)).In lymphocytes, glycolytic metabolism may play a critical role incontrolling cell survival. Withdrawal of exogenous survival factorsresults in a decline in cellular ATP, which is due, in part, todecreased expression of Glut-1, the major glucose transporter inlymphocytes (Lee et al, Nat. Med. 5:677-685 (1999)). Major cell survivalpathways have been reported to alter the metabolic response oflymphocytes to withdrawal of survival factors; sustain ATP production inmitochondria; increase glucose uptake; and/or enhance glycolysis in theabsence of extracellular signals (Lee et al, Nat. Med. 5:677-685 (1999),Vander Heiden et al, Mol. Cell 3:159-167 (1999)). CD8⁺ T-cell activationis accompanied by a dramatic increase in glucose uptake throughupregulation of Glut-1. It has been recently reported that CD3/CD28T-cell costimulation increases glycolytic flux, in a manner similar tothat of the insulin receptor (Frauwirth et al, Immunity 16:769-777(2002)). Letal also induced a dramatic increase in glucose uptake andup-regulation of Glut-1. Thus, NKG2D engagement, similarly to CD3/CD28costimulation, allows T-cells to anticipate the energetic needs of asustained immune response and appears to afford pro-survival signalsthrough regulation of the glycolytic pathway. Interestingly, Letalsignaling alone could trigger glucose up-take, thus the parallels anddifferences with the CD28 pathway remain to be established. Importantquestions follow on the mechanisms accounting for failure ofimmunosurveillance. It is possible that surveillance eventually selectsfor immunoresistant tumor variants that are capable of escapingCTL-mediated killing, inducing T-cell apoptosis or unresponsiveness(anergy) (Boon and van Baren, N. Engl. J. Med. 348:252-254 (2003)), orsimply dividing faster than CTL can kill. Letal could be then used as acostimulatory ligand to expand ex vivo in a CD28-independent mannerapoptosis-resistant tumor reactive T-cells for adoptive transfer usingartificial APCs. Given that peripheral effector CD8⁺ cells are mainlyCD28^(low/neg), such approach might offer significant advantage overCD28-based costimulation. The fact that anti-tumor response variesdepending on the level of NKG2D ligands that are expressed (Diefenbachet al, Nature 413:165-171 (2001)) supports the notion that expansion ofspecific T-cells at tumor sites, or protection of them againstchemotherapy, can be boosted by engineering cells with higher levels ofLetal or using soluble forms of the ligand.

All documents cited above are hereby incorporated in their entirety byreference.

1. A polypeptide comprising the sequence of SEQ ID NO:8, or variant orfragment thereof.
 2. The polypeptide according to claim 1 wherein saidpolypeptide comprises the sequence of SEQ ID NO:8 or variant thereofthat shares at least 50% identity with the sequence of SEQ ID NO:8. 3.The polypeptide according to claim 2 wherein said variant shares atleast 70% identity with the sequence of SEQ ID NO:8.
 4. The polypeptideaccording to claim 3 wherein said variant shares at least 90% identitywith the sequence of SEQ ID NO:8.
 5. The polypeptide according to claim1 wherein said polypeptide comprises the sequence of SEQ ID NO:8 orfragment thereof of at least 5 contiguous amino acids.
 6. Thepolypeptide according to claim 5 wherein said polypeptide comprises thesequence of SEQ ID NO:8 or fragment thereof of at least 20 contiguousamino acids.
 7. A polypeptide comprising at least one of the signalpeptide, the α-1 domain, the α-2 domain, the transmembrane domain andthe cytoplasmic domain of the sequence of SEQ ID NO:8 or variantthereof.
 8. The polypeptide according to claim 7 wherein saidpolypeptide comprises about amino acid 29 to about amino acid 225 of thesequence of SEQ ID NO:8.
 9. An isolated nucleic acid that encodes thepolypeptide according to claim 1, or a nucleic acid complementarythereto.
 10. The nucleic acid according to claim 9 wherein said nucleicacid comprises the sequence of nucleotides shown in SEQ ID NOs:1-4 thatencode the sequence of SEQ ID NO:8, or a nucleic acid complementarythereto.
 11. The nucleic acid according to claim 9 wherein said nucleicacid comprises a nucleotide sequence sharing at least 50% identity withthe nucleotide sequence set forth in SEQ ID NOs:1-4 that encodes theamino acid sequence set forth in SEQ ID NO:8.
 12. A construct comprisinga vector and the nucleic acid according to claim
 9. 13. The constructaccording to claim 12 wherein said vector is a viral vector.
 14. Theconstruct according to claim 12 wherein said nucleic acid is operablylinked to a promoter.
 15. The construct according to claim 14 whereinsaid promoter is a tumor specific promoter.
 16. A host cell comprisingthe construct according to claim
 12. 17. The host cell according toclaim 16 wherein said host cell is a mammalian cell.
 18. A method ofproducing a polypeptide comprising culturing the host cell according toclaim 16 under conditions such that said nucleic acid is expressed andsaid polypeptide is thereby produced.
 19. A therapeutic methodcomprising administering to a patient in need thereof the polypeptideaccording to claim 1 in an amount sufficient to stimulate effectorimmune cells of said patient.
 20. The method according to claim 19wherein said patient bears a tumor.
 21. The method according to claim 20wherein a nucleic acid encoding said polypeptide according to claim 1 isintroduced into tumor cells of said patient.
 22. The method according toclaim 19 wherein said patient has a viral infection.
 23. An antibodyspecific for the polypeptide of claim 1, or binding fragment thereof.24. A polypeptide comprising the sequence of SEQ ID NO:10, or variant orfragment thereof.
 25. A polypeptide comprising at least one of thetransmembrane, cytoplasmic and extracellular domains of the sequence ofSEQ ID NO:10 or variant thereof.
 26. An isolated nucleic acid thatencodes the polypeptide according to claim 24, or a nucleic acidcomplementary thereto.
 27. A construct comprising a vector in thenucleic acid according to claim
 26. 28. A host cell comprising theconstruct according to claim
 27. 29. A method of producing a polypeptidecomprising culturing the host cell according to claim 28 underconditions such that said nucleic acid is expressed and said polypeptideis thereby produced.
 30. An antibody specific for the polypeptide ofclaim 24.